Homogeneous detection of nucleic acid sequences is well known. Detection may include a dye, for example SYBR Green that fluoresces in the presence of double-stranded amplification reaction product or a fluorescently labeled oligonucleotide hybridization probe. For hybridization probes, “homogeneous detection” means detection that does not require separation of bound (hybridized to target) probes from unbound probes. Among probes suitable for homogeneous detection are dual-labeled probes (e.g., single stranded probes comprising a labeling moiety (e.g., fluorophore and/or quencher) at both the 5′ and 3′ ends), comprised of single-stranded oligonucleotides with a covalently bound fluorophore on one end and covalently bound quencher on the other end (e.g., quenched probe or self-quenching probed) whose absorption spectrum substantially overlaps the fluorophore's emission spectrum for FRET quenching when the probe is not bound to a target (5′ exonuclease probes described in, for example, Livak et al. (1995) PCR Methods Appl. 4:357-362; herein incorporated by reference in its entirety), hairpin probes labeled on one end with a fluorophore and on the other end with a quencher (molecular beacon probes described in, for example, Tyagi et al. (1996) Nature Biotechnology 14:303-308; herein incorporated by reference in its entirety). At an appropriate temperature a hairpin probe has a stem/loop structure when not bound to a target and in this structure the fluorophore and the quencher interact so closely that they engage in contact-quenching rather than FRET quenching. Double-stranded probes can also be used in homogeneous reactions. These probes have a covalently linked fluorophore on one strand and a covalently linked quencher on the complementary end of the other strand (yin-yang probes described in, for example, Li et al. (2002) Nucl. Acids Res. 30, No. 2 e5; herein incorporated by reference in its entirety), in addition linear probes having a fluorophore that absorbs emission from a fluorophore and re-emits at a longer wavelength (probes described in, for example, United States published patent application US2002/0110450; herein incorporated by reference in its entirety), and pairs of linear probes, one labeled with a donor fluorophore and one labeled with an acceptor fluorophore that hybridize near to one another on a target strand such that their labels interact by FRET (FRET probe pairs described in, for example, U.S. Pat. No. 6,140,054; herein incorporated by reference in its entirety). Detection methods include methods for detecting probes bound to single-stranded nucleic acid sequences (including variant target sequences), double-stranded targets including heteroduplexes comprised of imperfect complementary strands, or both.
Nucleic acid target sequences suitable for probing can be obtained directly in some instances by isolation and purification of nucleic acid in a sample. In other instances nucleic acid amplification is required. Amplification methods for use with homogeneous detection include the polymerase chain reaction (PCR), including symmetric PCR, asymmetric PCR and LATE-PCR, any of which can be combined with reverse transcription for amplifying RNA sequences, NASBA, SDA, and rolling circle amplification. Amplification-detection methods may rely on fluorescence due to probe hybridization, or they may rely on digestion of hybridized probes during amplification, for example, the 5′ nuclease amplification-detection method. If a sample contains or is amplified to contain, double-stranded target, for example, the amplification product of a symmetric PCR reaction, but single-stranded target is desired, separation of plus and minus strands can be accomplished by known methods, for example, by labeling one primer with biotin and separating the biotin-containing product strands from the other strands by capture onto an avidin-containing surface, which is then washed.
Certain fluorescent probes useful for homogeneous detection contain a fluorophore-labeled strand that emits a detectable signal when it hybridizes to its target sequence in a sample. For example, a molecular beacon probe is single-stranded and emits a detectable fluorescent signal upon hybridization. A ResonSense® probe is also single stranded and signals only when hybridized provided that the sample contains a dye, generally a SYBR dye, which stimulates hybridized probes by FRET when the dye is stimulated. Yin-yang probes are quenched double-stranded probes that include a fluorophore-labeled strand that emits a detectable signal it hybridizes to its target. FRET probe pairs, on the other hand, are probe pairs that emit a detectable fluorescent signal when both probes of the pair hybridize to their target sequences. Some amplification assays, notably the 5′ nuclease assay, include signal generation caused by probe cutting to generate fluorescent probe fragments rather than simply probe hybridization.
Certain probes that generate a signal upon hybridization can be constructed so as to be “allele-specific,” that is, to hybridize only to perfectly complementary target sequences, or to be mismatch-tolerant, that is, to hybridize to target sequences that either are perfectly complementary to the probe sequence or, hybridize at a somewhat lower temperature to target sequences that are generally complementary but contain one or more mismatches. Allele-specific molecular beacon probes have relatively short probe sequences, generally single-stranded loops not more than 25 nucleotides long with hairpin stems 4-6 nucleotides long, and are useful to detect, for example, single-nucleotide polymorphisms. Marras et al. (1999) Genetic Analysis Biomolecular Engineering 14: 151-156 (herein incorporated by reference in its entirety), discloses a real-time symmetric PCR assay that includes in the reaction mixture four molecular beacons having 16-nucleotide long probe sequences and 5-nucleotide stems, wherein each probe is a different color, that is, includes a fluorophore that is detectably distinguishable by its emission wavelength, and a probe sequence differing from the others by a single nucleotide. The sample is analyzed after each PCR cycle to detect which color arises and thereby to identify which of four possible target sequences perfectly complementary to one of the probes is present in a sample. Mismatch-tolerant molecular beacon probes have longer probe sequences, generally single-stranded loops of up to 50 or even 60 nucleotides with hairpin stems maintained at 4-7 nucleotides. Tyagi et al. European Patent No. 1230387 (herein incorporated by reference in its entirety) discloses a symmetric PCR amplification and homogeneous detection assay using a set of four differently colored mismatch-tolerant molecular beacon probes having different probe sequences 40-45 nucleotides long and stems 5-7 nucleotides long, to hybridize competitively to, and thereby interrogate, a 42-nucleotide long hypervariable sequence of mycobacterial 16S rRNA genes to determine which of eight mycobacterial species is present in a sample. The sample is analyzed by determining a ratio of fluorophore intensities at one or more temperatures to identify the species that is present. El-Hajj et al (2009) J. Clin. Microbiology 47:1190-1198 (herein incorporated by reference in its entirety), discloses a LATE-PCR amplification and homogeneous detection assay similarly using four differently colored mismatch-tolerant molecular beacon probes having different probe sequences 36-39 nucleotides long and stems 5 nucleotides long to hybridize competitively to, and thereby interrogate, a 39-nucleotide long hypervariable sequence of mycobacterial 16S rRNA genes to determine which of twenty-seven mycobacterial species is present in a sample. Each of the four probes is a “consensus probe,” that is, it has a single-stranded loop complementary to multiple species but perfectly complementary to none of them. Genomic DNA from some 27 different species were separately amplified, the Tm of each probe was determined by post-amplification melt analysis, and data was tabulated. To analyze a sample containing an unknown species, the sample was amplified and analyzed as above. The Tm's of all four probes were compared to the tabulated results to identify the species present in the sample.
Multiple probes, both mismatch-tolerant and allele-specific, have been used to interrogate multiple target sequences. El-Hajj et al. (2001) J. Clin. Microbiology 39:4131-4137 (herein incorporated by reference in its entirety), discloses performing a single, multiplex, real-time, symmetric PCR assay containing five differently colored, allele-specific molecular beacons, three complementary to one amplicon strand and two complementary to the other amplicon strand, which together span an 81-nucleotide long region of the rpoB gene core region of M. tuberculosis in overlapping fashion. Probe fluorescence intensities were obtained, and failure of any one of the probes to hybridize and signal was taken as an indication of drug resistance. Wittwer et al. U.S. Pat. No. 6,140,054 (herein incorporated by reference in its entirety) discloses a multiplex symmetric PCR assay for detecting single and double base-pair mismatches in two sequences (C282Y and H63D sites) of the human HFE gene using a primer pair for each site, a FRET probe pair for each site, and rapid thermal cycling. Each probe pair includes a mismatch-tolerant fluorescein donor probe 20-30 nucleotides in length, positioned to hybridize to target sites of possible variations, and a Cy5 acceptor probe 35-45 nucleotides long, called the “anchor” probe, because it remains hybridized as its companion fluorescein probe melts off the target sequence at a melting temperature dependent on its degree of complementarity.
Usable single-tube assays that provide detailed characterization of multiple target sequences using a single-color reporter would reduce the cost and complexity analysis.